With the advances on gene therapy technologies, viral vectors are applied more and more in treating human diseases. Among them, lentiviral vectors is the commonly used viral vectors. The lentiviral vector is a reconstructed viral vector system on the basis of HIV-1 virus and can introduce the gene of interest efficiently into animal and human primary cells or cell-lines. The genome of the lentiviral vector which is a plus-strand RNA enters into the cell and is reversely transcribed into DNA in the cytoplasm by a reverse-transcriptase carried by the genome itself to form a DNA pre-integration complex. Then, the complex is transported into the cell nuclei and the DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNAs which are returned to the cytoplasm and expressed into proteins of interest.
The lentiviral vector-mediated gene expression has continuous and stable effect, because the gene of interest has been integrated into a host cell's genome and is divided with the division of the cell genome. Moreover, the lentiviral vector can effectively infect a nondividing cell and can be effectively integrated into the nondividing cell. With the above-mentioned properties, the lentiviral vector has great advantages as compared to the other viral vectors, such as non-integrated adenoviral vector, adeno-associated viral vector with low integration rate, classical retroviral vector which is solely integrated into a dividing cell. Today, tissues or cells where the lentiviral vector-mediated chronic expression of a gene of interest can occur include brain, liver, muscular, retina, hematopoietic stem cells, marrow mesenchymal stem cells, macrophages, etc.
Nevertheless, the stability of the lentiviral vector is very low, while a viral vector which will be used in the therapeutic application of the human body must keep the structural integrity to maintain its biological activity. In order to keep the structural integrity of the viral vector to maintain its biological activity, such preparations must be maintained and delivered at a relatively low temperature.
Loss of biological activities of the lentiviral vector mostly occurs at storage stage. A recombinant lentiviral vector carrying a gene of interest is typically prepared into a liquid preparation and stored at an ultra-low temperature (e.g. no higher than −60° C.), transported under the condition of low temperature and frozen, and thawed before use. It is one of the major challenges that the structural and functional ingredients are prevented from being physically disrupted during freezing and storing at a temperature below freezing point.
Typically, the lentiviral vector preparations comprise proteins encoded by the viral genome and a single- or double-stranded genome. If the virus contains an envelope protein, the preparation further comprises a lipid two-layer membrane. Under a ultra-low temperature, the protein is liable to denaturation and the lipid two-layer membrane is liable to destruction during freezing. Therefore, it is inappropriate for a viral vector preparation to be stored under an ultra-low temperature for a long time.
Furthermore, when the recombinant lentiviral vector is stored at an ultra-low temperature for too long time (e.g., longer than 200 days), when the recombinant lentiviral vector is under freeze-thaw cycles during administration, or when the recombinant lentiviral vector is exposed to the body temperature of an organism (e.g. body temperature of the human being or other animals) or the room temperature too longer time before the preparation reaches the target organ due to improper application, the biological activity of the recombinant lentiviral vector (typically expressed in virus titer) often decreases quickly and dramatically, which directly influences on therapeutic effect of the recombinant lentiviral vector preparation or leads to inaccuracy in results of preclinical or clinical study.
Furthermore, for a clinical setting short of suitable storage apparatuses for viral vectors, the hospital's willingness to adopt the virus preparation depends on the using cost of a virus preparation. Generally, pharmacies and hospitals have freezing apparatuses with the lowest temperature of −20° C., but hardly have freezing apparatuses with the lowest temperature of −80° C. It is always believed that a liquid preparation of a viral vector will be stable only at an ultra-low temperature.
Therefore, there is an urgent need in a recombinant lentiviral vector preparation with good stability. The recombinant lentiviral vector preparation can be stored for a long time under the condition of an ultra-low temperature of −80° C., or can also keep long-term stability under the condition of a middle- or low-temperature of −20° C., or can still be capable of maintaining the activities of the recombinant lentiviral vector which can meet to the requirement in use, after freeze-thaw cycles during administration or after exposure to an organism's body temperature or the room temperature for a longer time.